Age-related macular degeneration (AMD) is a retinal degenerative disease that causes progressive loss of central vision. The risk of developing AMD increases with age and most often affects people in their sixties and seventies or older. AMD is the leading cause of legal blindness in the developed world in patients over the age of sixty-five, and it ranks second, after diabetic retinopathy, between age 45 and 65.
Central vision loss from AMD is caused by photoreceptor degeneration in the macula region. The macula region is the central portion of the fundus and retina, responsible for perceiving fine visual details and the macula region has the highest photoreceptor density of the eye. The fundus is the posterior ½-⅓ part of the eye and contains the same three tissue layers as the rest of the eye, retina, chorioid and sclera (anterior to posterior). Light sensing cells in the retina, known as photoreceptor cells, convert light into electrical impulses and then transfer these impulses to the brain via the optic nerve.
The following abbreviations are used throughout the text:
AMDAge-related macular degenerationCCDCharge coupled deviceCNVChoroidal neovascularizationICGIndocyanine greenNIRNear infraredPDTPhotodynamic therapyRGDArginine-glycine-aspartic acidRPERetinal pigment epitheliumSLOScanning laser ophthalmoscopyVEGFVascular endothelial growth factor
There are two types of AMD: dry and wet. Dry AMD is also called atrophic, nonexudative, or drusenoid macular degeneration. With dry AMD, yellow-white deposits called drusen accumulate between the retinal pigment epithelium (RPE) tissue and Bruch's membrane as a result of accumulated retinal waste. Drusen deposits are visible in white and fluorescent light fundus imaging and no contrast agent is required for diagnosis. Drusen deposits are composed of waste products from photoreceptor cells, which are not handled correctly by the RPE. The RPE cells normally act as “gatekeepers”, supplying nutrients to and scavenging waste products from the photoreceptors across Bruch's membrane. The drusen deposits, which occur in as well as outside the macula region, are thought to interfere with the normal function and maintenance of photoreceptors, causing progressive degeneration of these cells. Drusen deposits can, however, be present in the retina for many years without vision loss. Vision loss from dry AMD occurs very gradually over the course of many years.
Wet AMD is characterised by the presence of choroidal neovascularization (CNV) and neovascular membranes, and is also called subretinal neovascularization, exudative, or disciform degeneration, in addition to CNV. In wet AMD, abnormal new blood vessels originate in the choroid and penetrate through Bruch's membrane. When these new or “angiogenic” vessels are located posterior to the RPE, a so called type I or “occult type” AMD is formed. When the new vessels are located anterior to the RPE, a so called type II or “classic type” AMD is formed. Combinations of type I and II also occur. These new vessels and the proliferative RPE response form “neovascular membranes”, which leak blood and fluid into the subretinal cells. The neovascular membranes are leaky because the fenestrations between the endothelial linings of angiogenic vessels are wider than in normal vessels and allow extravasation of macromolecular substances (plasma and blood). The process of new vessel formation, as well as the blood and fluid leakage, damage the photoreceptor cells and further compromise the RPE support of the photoreceptors. Current methods of AMD diagnosis with fluorescein and indocyanine green (ICG) rely on the leaky nature of angiogenic vessels and hence focal accumulation of the fluorophores due to this morphological/structural abnormality. Diagnosis of AMD is therefore today only possible at relatively advanced stages of AMD, when sufficient neovascular membranes have formed. Due to the relatively late diagnosis with the diagnostic agents available today, wet AMD therefore tends to progress rapidly and carries the highest risk of severe impairment to vision. In a majority of patients with wet AMD occult lesions are present particularly during the initial stages of AMD. Despite the above knowledge about AMD pathology, many aspects and mechanism of CNV are not well understood.
In the USA today there are about 15 million people having AMD, 90% having the dry type and 10% having the wet type. It has been estimated that there will be 2 million new cases per year in the USA in the future.
Generally, new blood vessels can be formed by two different mechanisms: vasculogenesis or angiogenesis. Angiogenesis is the formation of new blood vessels by branching from existing vessels and occurs as a normal event in embryologic development and health, as well as part of various disease processes. The primary stimulus for this process may be inadequate supply of nutrients and oxygen (hypoxia) to cells in a tissue. The cells may respond by secreting angiogenic factors, of which there are many; one example, which is frequently referred to, is vascular endothelial growth factor (VEGF). These factors initiate the secretion of proteolytic enzymes that break down the proteins of the basement membrane, as well as inhibitors that limit the action of these potentially harmful enzymes. The other prominent effect of angiogenic factors is to cause endothelial cells to migrate and divide. Endothelial cells that are attached to the basement membrane do not undergo mitosis. The combined effect of loss of attachment and signals from the receptors for angiogenic factors is to cause the endothelial cells to move, multiply, and rearrange themselves, and finally to synthesise a basement membrane around the new vessels.
Angiogenesis involves receptors that are unique to endothelial cells and surrounding tissues. These angiogenesis receptors include growth factor receptors such as VEGF and the integrin receptors. Immunohistochemical studies have demonstrated that a variety of integrins, perhaps most importantly the αv class, are expressed on the apical surface of blood vessels [Conforti, G., et al. (1992) Blood 80: 37-446] and are available for targeting by circulating ligands [Pasqualini, R., et al. (1997) Nature Biotechnology 15: 542-546].
The integrins αvβ3 and αvβ5 are receptors known to be associated with angiogenesis. Stimulated endothelial cells appear to rely on these receptors for survival during a critical period of the angiogeneic process, as antagonists of the αvβ3 integrin receptor/ligand interaction induce apoptosis and inhibit blood vessel growth.
Integrins are heterodimeric molecules in which the α- and β-subunits penetrate the cell-membrane lipid bilayer. The α-subunit has four Ca2+ binding domains on its extracellular chain, and the β-subunit has a number of extracellular cysteine-rich domains.
Many ligands (e.g. fibronectin) involved in cell adhesion contain the tripeptide sequence arginine-glycine-aspartic acid (RGD). The RGD sequence appears to act as a primary recognition site between the ligands presenting this sequence and receptors on the surface of cells. It is generally believed that secondary interactions between the ligand and receptor enhance the specificity of the interaction. These secondary interactions might take place between moieties of the ligand and receptor that are immediately adjacent to the RGD sequence or at sites that are distant from the RGD sequence.
RGD peptides are known to bind to a range of integrin receptors and have the potential to regulate a number of cellular events of significant application in the clinical setting. Perhaps the most widely studied effect of RGD peptides and mimetics thereof relate to their use as anti-thrombotic agents where they target the platelet integrin GpIIbIIIa.
Examples of RGD-containing peptide-based contrast agents are found in WO 01/77145, WO 02/26776 and WO 03/006491.
In some cases, if wet AMD is diagnosed early, laser surgery (photocoagulation) can prevent extensive central vision loss. In this type of surgery, laser beams cauterise the leaky blood vessels of the neovascular membranes. For laser surgery to be effective, it is critical that wet AMD is diagnosed before extensive vision loss occurs.
Another treatment for wet AMD is photodynamic therapy (PDT). PDT uses a light sensitive drug (e.g. Visudyne™; liposomal BPD-MA verteporfin) in combination with laser treatment. Administered intravenously, the photosensitive drug is inactive and accumulates in the undesired neovascular membranes. A predetermined amount of low-energy laser light is delivered to the target CNV tissue, which activates the drug contained within the vessels. The laser light, when combined with the photosensitive drug, invokes a highly reactive form of oxygen in the target tissue that leads to closure of the unwanted blood vessels. PDT therapy is highly selective and involves low-level, non-thermal laser energy, minimizing damage to the surrounding retinal tissue. The entire procedure can be accomplished in approximately 30 minutes and is performed on an outpatient basis. Typically, the new blood vessels remain closed for a few weeks or months, after which a re-treatment may be necessary. There is hence a need for methods to diagnose wet AMD and to monitor the treatment of the indication.
The existing methods for detection of wet AMD is by angiography with a fundus camera during intravenous administration of fluorescein and/or ICG. The diagnostic principle for both agents is detection of vascular morphological/structural changes, leading to leakage from angiogenic vessels and requiring relatively high dosages to obtain bolus passage and sufficient tissue concentrations after systemic distribution. After a short arterial and venous bolus phase upon intravenous administration, fluorescein extravasates and distributes relatively rapid in the extracellular fluid space. A diffuse and dominating fluorescence signal from the choroidal vasculature is therefore observed from 20-30 seconds after administration of fluorescein (without any anatomical resolution). Angiogenic vessels are hence distinguished by “excessive” neovascular membranes and present as locally increased fluorescence. Leakage of fluorescein in classic type AMD is easier to diagnose (located anterior to the RPE) and shows well-demarcated areas of hyperfluorescence and homogeneous leakage. As indicated by its name, occult type AMD is more difficult to diagnose with fluorescein, as the RPE act as a very effective optical barrier to both excitation and emission light. Use of fluorescein is therefore not reliable for occult type AMD, although irregular fluorescence (“stippling”) in the early phase and inhomogeneous leakage at the late phase is observed in some cases. Analysis of fluorescein angiograms can yield haemodynamic information such as arteriovenous passage time and mean dye velocity. U.S. Pat. No. 6,599,891 is one of many examples disclosing methods of PDT and imaging using fluorescein angiography.
ICG binds very strongly to albumin and hence behaves more or less like a blood pool tracer. The maximum excitation and emission wave lengths of ICG are 806 nm and 830 nm, respectively, resulting in relatively little absorption by the RPE. The RPE light absorption is dependent upon the wavelength and is the least in the near-infrared range. ICG does hence confer angiographic details from the choroidal tissue and tissues posterior to the RPE layer, which is of importance in cases of occult type AMD. However, as with fluorescein, vascular morphological/structural changes and passive leakage is the diagnostic principle with ICG and only well advanced focal neovascular membranes can therefore be diagnosed with ICG. US2004/0156782 discloses a method of using ICG for obtaining angiographic images of CNV.
Neither fluorescein nor ICG angiography provide methods for detection of wet AMD in early phases, before substantial volumes of neovascular membranes have formed and visual impairment has started.
WO 2004/034889 discloses methods, apparatus and systems for the photodynamic treatment of feeder vessels associated with choroidal neovasculature, and also describes the need for detection of such feeder vessels. The therapy method includes using a photosensitizer with may be coupled to a binding ligand which binds to a specific surface component of the target ocular tissue. To detect the feeder vessels conventional fluoroescein angiography and ICG angiography are suggested used.
There is a clinical need to develop more specific non-invasive imaging techniques for AMD, and particularly for wet AMD and occult choroidal neovascularisation. The existing techniques do not diagnose the underlying pathology, but only identify morpholocal/structural changes by leaky angiogenic vessels when the formation and volume of neovascular membranes is well advanced. As the relatively poor prognosis of wet AMD is among other due to the relatively late diagnosis provided with the currently available methods, new imaging methods with the ability to diagnose smaller and earlier lesions and preferably the nature of the underlying pathology will be of clinical benefit in diagnosing and monitoring the development of AMD at an early stage. Such imaging methods will also have a central role in the evaluation of novel anti-angiogenic therapies.